Practical (Soil and

Aquatic Microbiology)

2020-2021

Ministry of Higher Education and Scientific Research

University of Baghdad College of Science

Department of Biology

د حلا مؤيد رديفا. م.

محمد صلاح الديه برهانأ.م.

شاكرخمائل لطفي أ.م.

أ.د. حُلا يووس فاضل جىان محمد حسهد. م.

شيماء فؤاد رشيد م.

اوتصار فرعون احمدم.

غافل . جىان عطيةم

Soil microbiology lab (1):

Soil microbiology

Soil is the material found on the surface of the earth that is composed of organic and inorganic material. Soil varies due to its structure and composition. Inorganic materials, or those materials that are not living, include weathered rocks and minerals. As rocks are broken down, they mix with organic materials, which are those materials that originate from living organisms. For example, plants and animals die and decompose, releasing nutrients back into the soil.

Types of Soil:

❖ there are three basic types of soil: sand, silt and clay. But, most soils are composed of a combination of the different types. How they mix will determine the texture of the soil, or, in other words, how the soil looks and feels.

Water content:

The water in the soil play a significant role, as it influences the metabolic activities of micro biota, soil water divided into two types:

1- Free water (Gravitational water) this water not bounded by colloidal material, which is affected by gravitation, so tend to drain quickly?

2- Bound water it’s a thin water film on soil particles surface, because it's bounded by colloidal material, this type of water not affected by gravitation, so tend to drain slowly?

Water activity (aw):

It's useful to express qualitatively the degree of water viability, microbiologist generally use aw, which is less amount of water required for microbial activities and growth, the water activity of a solution is 1/100 the humidity of a solution (expressed as a percentage). Water activity of a solution or solid can be determined by different methods, the most current assay method is:

Drying method

Procedure:

1. Weight 10gm of soil in previously weighted clean Petri dish.

2. Dehydrate soil in hot air oven at 105° C for 3-4 hrs.

3. Reweight dried soil sample, and determine (aw) of sample according to the following.

Water content of soil sample = weight of moist soil - weight of dry soil

Weight of dry soil sample =l 0 - water content

Weight of soil sample for 1 gm dry soil = weight of dry soil

weight of moist soil

Enumeration of soil Microorganisms

Soil microorganisms can be classified as: Bacteria, Actinomycetes, fungi, Algae and protozoa. Each of these groups has characteristics that define them and their functions in soil. Fertile soil contains a wide variety of M.O. but there is no particular isolation lab. Procedure can be giving a quiet and accurate microbial numbers in a soil sample?

Enumeration methods:

1. Direct slide count (Breed Method) This method considered the quickest and simplest technique, although there are some disadvantages like:

▪ Estimate number of live and dead cells in sample.

▪ Bacteria are dominant in soil so it's difficult to distinguish other microbial cells that are present, such as microbial spores.

▪ can’t distinguish microbes from soil particles.

Procedure:

1. Mark square (1 cm diameters) on clean glass slide. 2. Suspend 1 gm of soil sample in 9 ml of sterile diluted water, mix properly. 3. Spread loop full of soil dilution (0.01) in the square.

4. Stain with simple stain. 5. Count visible microbial cells in 10 microscopic fields and determine average no. of bacteria in soil sample from equation:

No. of cells = average of cells in 10 fields x5000x100 x 10

▪ To find the No. of cells in dried wt. of soil sample?

No. of cells in 1 gm of dry soil sample= No. of bacterial cells

dry wt of soil sample

Constant factors:

5000 = no. of fields in 1 cm2

100 = (0.01) conversion of loop full volume to 1 ml.

10 = reciprocal of dilution

2 - Dilution plate count (Viable Plate Count)

This technique is widely used for determining approximate viable no. of soil bacteria, and it's applicable to any M.O. that will grow as colonies, but this method have some disadvantages:

▪ It ignores sampling error due to unequal distribution of the cells, especially in the high dilutions.

▪ There is no ideal culture medium for cultivation of most soil bacteria.

▪ Antagonistic activities of soil bacteria, such as production of antibiotics and some enzymes that inhibit the growth of another M.O.

No of cell / gm soil = No. of viable colonies x dilution factor x ?

No of cell / gm of dried soil = No. of cell

Wt. of 1gm dry soil

Lab (2):

Isolation of soil microorganisms

The most numerous microbes in soil are the bacteria followed by actinomycetes, the

fungi, soil algae and soil protozoa but in our study we are going to concentrate on bacteria

,actinomycetes and fungi .

Isolation of Bacteria

We are going to isolate bacteria from soil by using Dilution plate count technique (Pouring plate

count).

First, we have to add 1 gm of soil to 9 ml D.W to get 10 -1

dilution, mix properly, farther dilutions

are depend on the soil type as the following:

• domestic soil 8 dilutions

• Vegetable soil 6 dilutions

• citrus soil 5 dilutions

• Palm soil 4 dilutions

• Uncultured soil 2 dilutions

• Then transfer 0.1 ml from each 3 last dilutions to sterile Petri dishes, after that pour melted

cold soil extract agar (pH 7) into dishes and thoroughly mix with still-fluid agar by a gentle

horizontal rotation of dish.

• Finally incubate plates at 30 °C for 24 hr.

Note: count plates which show only about 30-300 colonies.

No of cell / gm soil = No. of viable colonies x dilution factor x ?

To find the No. of bacterial cells in dried wt. of soil sample

No of cell / gm of dried soil = No. of cell

Wt. of 1gm dry soil

Note: For isolation and enumeration of spore forming soil bacteria (heat the last sample

dilution at 80 ° C water bath for about 15 min), then transfer 0.1 ml to Petri dish and

continue the same procedure as above.

Lab (3): Isolation of Soil Fungi

Fungi typically divided into:

1- Molds: which are composed of branching filaments termed hyphae, that grow by apical

extension to form mycelium. They are helpful but could also be harmful to plant and other soil

microorganism.

2- Yeasts: which are unicellular and oval or round in shape and reproduce mainly a sexually by

budding.

Isolation Procedure 1- Add 1 gm of soil sample to 9 ml D.W., and then make 10-fold serial dilution by D.W.

2- Transfer 1 ml from the last dilution to Petri-dish, then pour melted Malt extract agar medium

(pH 5.5) or Rosebengal agar medium (pH 6) and thoroughly mix.

3- Incubate plates at 28 C0 for week.

4- Examine formed colonies and recognize their color and morphology.

Slide preparation for mold Add one drop of lactophenol in the middle of the slide , take apportion of colony , put cover slip,

gently press the cover edge by loop and examine microscopically under high power.

• Slide preparation for yeast

• Simple stain.

3. Isolation of Actinomyces

Actinomycetes are a group of gram- positive bacteria which form branched filamentous

hyphae like fungal hyphae but their hyphal diameter is less than fungal hyphae.

Actinomycetes give soil its characteristic smell due to their production of volatile substances

such as geosmin, Actinomyces prefer slightly alkaline environments.

Actinomycetes colonies are powdery mass form often pigmented with orange or red or yellow

color.

Isolation procedure 1- Add1 gm of soil sample to 9 ml of D.W., and then makes serial 10-fold dilution with D.W.

2- Spread 1 ml of last dilution on Jensen's agar plates, pH8.5-9 (Actinomycetes selective

medium), incubate at 280C for 3-7 days.

3- prepare slide using gram procedure, examine under high power, and observe Actinomycetes

thin curly mycelia net.

Lab (4):

Role of Soil Microbes in Elements Recycling

Human & other organisms cells chemical composition can be described as a complex blend

of organic compounds consist of different elements such as C , N , O and H in variety

combinations , these elements which are essential component of all organism protoplasm

undergo cyclical alteration between inorganic state free in nature and a combined organic

state in living organisms .

Microorganisms play a major role in degradation (decomposition) of organic matter, that

contribute in elements recycling in which maintain their balance & keep life on the earth

from ceasing.

Carbon Cycle

The element carbon is the present in all living organisms. It's recycled through various

processes, Transformation of carbon occurs constantly and ubiquitously, carbon is

introduced into organic system from its most oxidized state Co2 & it reduced primarily by

photosynthesis and become part of photosynthetic organism's components as organic carbon

in that temporarily. Immobilized until decomposition of cells by M.O.

Role of soil Micro Organisms in degradation of carbohydrates 1-

Degradation of cellulose

Cellulose….. Is a complex carbohydrate, which is part of plant structure polysaccharides,

soil contain a rich deposit of M.O which produce extracellular cellulolase that break down

cellulose into two to three glucose units called cellobiose and cellotriose respectively. These

smaller compounds are readily degraded and assimilated as glucose.

Bacteria Cytophaga Cellulomonas

Fungi Aspergillus Penicilluim Trichoderma

Actionomycetes Nocardia

Isolation of Soil Cellulytic Microorganism

Procedure:

1- Suspend 1 gm of soil sample in 9 ml of special isolation broth (the medium contains

all required nutrients except carbon source?)

2- Transfer 1 ml of soil suspension to other tube contain the same isolation medium + a

strip of filter paper (as carbon source). Incubated tubes at 280C for 5 – 7days.

4- +ve result for cellulytic m.o yellow spots on filter paper strips (The cellulytic bacterial

colonies colored by yellow to orange color due to formation of carotenoid or flexiratin

pigments).

5- - ve result no color on the strip.

6- Prepare smear of detected spots on glass slide, stain with gram stain & examine under oil

immersion.

2- Degradation of Pectin

Pectin….. is a homopolymer of D-galactronic acid found in middle lamella of plant cell

wall & in blue green algae capsules .( in plant pectin solidify & support plant cells by their

combination with calcium carbonate) .

Microbial pectinolytic enzymes (pectinase) are responsible for the lyses of pectin, which

enable M.O to invade tissues of living plants causing soft rot as well as wet or dry necrosis

& galls in economically important crops such as potatoes, carrot and cucumber.

Bacteria Erwinia

Fungi Fusarium

Isolation of soil Pectinolytic Microorganism

Procedure

1- Suspend 1 gm of soil sample in 9 ml of special isolation broth medium. 2- Transfer

1 ml of soil suspension to other tube contain the same isolation medium with piece of

potato (as source of carbon), incubate at 28 0C for 5 – 7 days.

3- + ve result color change and soften of potato.

4- Prepare slide, stain with gram stain and examine under oil immersion.

3- Degradation of starch

Starch….. Consist of long chain of ∝ - glucosyl residues, Starch break down by amylases,

which produced by a variety of living organisms, ranging from bacteria to plants & human.

Bacteria & fungi secrete amylase to outside of their cells to carry out

extracellular digestion, where they have broken down the soluble and products such as

glucose and maltose are absorbed into their cells.

Amylases are classified based on how they break down starch molecules.

α- Amylase (alpha–amylase) : reduce the viscosity of starch by breaks the glucose glucose bonds

down by removing two glucose units at a time therefore producing of maltose.

β- Amylase (Beta-amylase) : breaking down the bonds at random way. therefore producing varies

size of glucose chains.

Amyloglucosides ( AMG) breaks successive bonds from the non-reducing ends of the straight

chain therefore producing of glucose.

Amylase producer Microorganism

Although many M.O produce these enzymes, but the most common producers

Bacteria Bacillus spp Clostridim spp Micrococcus

Fungi Aspergillus Fusarium Rhizopus

Isolation of soil amylase producers Microorganisms

Procedure

1- Suspend 1 gm of soil sample in 9 ml of special isolation broth medium for isolation

of starch hydrolysis M.O.

2- Incubate tubes at 28 C0for a week.

3- Transfer adequate volume of the medium to a test tube & add drops of gram's iodine

solution.

4- + ve result yellow color confirm positive result, due to microbial hydrolysis of starch,

- ve result blue color confirm negative result .

Another detection procedure

For isolation of amylolytic fungi….. Spread 1 ml of soil suspension on potato dextrose agar

plates PDA (with 0.1 mg/ml streptomycin sulfate), incubate at R.T for about 3-7 days.

For isolation of soil amylolytic bacteria….. Spread 1 ml of soil suspension on nutrient agar plate

(1 % w/v soluble starch) at 300 C for 24 hr.

Starch hydrolyzing colonies will have an area of clearing around them, for confirming, flood plates

with gram's iodine (area around the colony appear yellow, while area still contain starch appear ink

– black color.

Lab (5): Nitrogen cycle

Is the biochemical cycle which nitrogen is converted into multiple chemical forms, consecutively

passing from the atmosphere to soil to organism and back into the atmosphere?

The main stages of nitrogen cycle are:

1. Ammonification

2. Nitrification

3. Denitrification or nitrate reduction

4. Nitrogen fixation

1. Ammonification

It is a series of enzyme reactions resulting in release of ammonia (NH3) from complex organic

nitrogenous compounds such as proteins and nucleic acids , usually occurs under aerobic

condition.

Numerous microorganisms can release ammonia from organic compounds in the soil such as:

Proteus and Micrococcus

Isolaion and detection of ammonification M.O.

Suspend 1 gm of the soil sample in 9 ml of Sodium casein broth medium (as source of

protein) then incubate tubes at 28 Co for a week.

Mix 1 ml microorganisms' suspension with few drops of Nessler reagent in clean test tube.

Positive result

Golden -orange deposit demonstrates releasing of ammonia

2. Nitrification

Oxidation of ammonia (produced from the degradation of organic compounds in

ammonification ) to nitrate by a specialized group of strictly aerobic

chemolithotrophes.

Oxidation of ammonia occurs in two steps: In the first step: ammonia is oxidized to nitrite (NO2

-) (nitrosofication)

The most involved microorganisms in this process are: Nitrosomonas and

Nitrosococcus

Isolation & detection of Nitrosofication M.O

1. Suspend 1 gm of the soil sample in 9 ml of Allen I broth medium (Contains

(NH4)2 SO4 as ammonia source), incubate tubes at 28 Co for a week.

2. Mix 1ml microbial suspension with an equal volume of reagent A and reagent B

let them react for a few seconds.

Positive result: Formation of a red colored deposit indicates releasing of NO2 as a

result of the nitrification process.

2. In the second step is oxidized NO2 to nitrate (NO-3),the Nitrobacter mainly carry out the

second step.

Isolation and detection Nitrite Oxidizing microorganisms

Follow procedure as in previous but use Allen II broth as a medium contains NaNO2 as nitrite

source. For detection of released NO3, mix 1 ml of microorganism's suspension with drops of

nitrate reagent which consists of Diphenylamine (DPA) and sulfuric acid (H2SO4) .

Positive result

Formation of blue deposit indicates releasing of No3.

Note: If deposit not observed, that does not mean absence of nitrifying microorganisms in taken

soil sample rather it mean ammonia oxidation continued.

3. Denitrification or nitrate reduction

Reduce nitrate to nitrite, ammonia and return to the atmosphere as nitrogen gas (N2↑), under

anaerobic conditions.

The reaction mediated by nitrate reductase enzyme that used nitrate as an electron acceptor in

anaerobic respiration.

This process is performed by bacterial species such as Pseudomonas and Clostridium .

Denitrification detection procedure

Suspend 1 gm of the soil sample in Allen l8 broth medium (the medium contain KNO3 as source

of NO3) in test tube. Fill tubes completely with medium to create anaerobic condition incubate at

28 Co for a week.

Result

Read the result by the following procedures:

• NO3 detection reagent: formation of blue deposit detects absence of denitrifying

microorganisms.

• NO2 detection reagent: formation of red colored deposit detects reduction of NO3 to NO2.

• Nessler reagent: formation of golden –orange deposit detects complete reduction of NO3 to

ammonia.

Lab (6): Nitrogen fixation

Is a process by which nitrogen gas is converted into ammonia or related nitrogenous

compounds in soil by soil microorganisms.

Nitrogenase is the most important enzyme involved in nitrogen fixation.

Two kinds of nitrogen-fixing microorganisms are recognized:

• Non symbiotic N2 fixer: Those are capable of converting N2 to cellular nitrogen

independently of other living organism’s so-called free-living nitrogen-fixing

bacteria.

• The main involved M.O. Azotobacter : large gram-negative motile rods that may

be ovoid or coccoidal in shape (pleomorphic), aerobic and cells form cysts as the

culture ages.

(Macroscopically)

Azotobacter has grown on solid agar media as large convex, mucoid colonies with

white, brownish color, colorless and any other color depend on bacterial species, as

soon as culture aged brownish colonies pigmented with dark chocolate color.

Isolation of Azotobacter

1. Suspend 1 gm of the soil sample in 9 ml of D.W.

2-Spread 1 ml of soil suspension on Ashby's agar medium pH 7.6, (selective Azotobacter

medium), includes sodium molybdenum, incubate the plates at 28C0 for a week.

Azotobacter characterized as mucoid and brownish large colonies.

Symbiotic N2 fixers Symbiotic N2 fixers microorganisms live in the roots of legume family plants and other

plants . the fixation process results from a mutualistic association between legumes plants

and bacteria.

main symbiotic N2 fixer bacteria is Rhizobium.

Which are G-ve rods to pleomorphic, motile with variably placed flagella.

Rhizobium invades susceptible plant roots and from visible nodules which live and fix N2

directly from the air.

Isolation of Rhizobium from soil

1. Cut roots of any legumes, wash with tap water.

2. Select and cut visible large pink nodule , transfer to clean glass slid, crush it carefully by

pressing it by forceps , until seeing a milky suspension then mix with water drop , stain with

gram stain , examine under oil immersion Rhizobium appears look like Latin letters Y X Z T ,

also bacterial can be detected as G-ve bands.

1

Aquatic Microbiology Lab (7):

Water is the elixir of life. It is an essential part of protoplasm and creates

a state for metabolic activities to occur smoothly. Therefore, there is no

life can exist without water. Also there are thousands of m.o. which live

in water so can do diseases.

Water receives m.o. from air, soil, sewage, organic wastes, dead plants

and animals…etc.

Water Microorganisms ….. large number of m.o. both saprophytes and

pathogens are found in water (bacteria, fungi, algae, protozoa, nematodes

several human and animal viruses) are transferred by water.

The natural water such as: lakes, streams, rivers, contain several amount

of nutrients that support the growth of m.o.

There are different ways by which m.o. enters water supply, for

example broken sewage lines, congested centers, inappropriate waste

treatment, lack of awareness among people , Unhygienic environments at

water public places, People suffering from diseases also discharge

pathogenic microbes in water through their excrete, for ex. amoeba

dysentery, typhoid fever, bacillary dysentery and poliomyelitis …etc.

Water Sanitary Tests

Feacal Bacterial Indicators

The water potability (suitability for drinking) is considered as one of the

basis in microbiological examination of water.

1- fecal coliforms Intestinal bacteria in water generally can’t survive in aquatic

environment due to physiological stress but if they can enter human in the

meanwhile, they can cause serious disease.

The characteristics groups of intestinal bacteria are the coliforms,

(facultative anaerobes, G-ve, non-spore former, rod , ferment lactose with

gas formation within 48hr at 30ºC).

The coliform groups are present in water due to fecal contamination i.e.

discharge of feces by human and animal in water. coliforms are members

of the family Enterobacteriaceae which includes: E.coli, Enterobacter,

salmonella and klebsiella pneumonia.

2

These bacteria make up about 10% of intestinal m.o. of human and

animal therefore was used as bacterial indicator of fecal water pollution.

If such bacteria cannot detected in water /100 ml, the water can

consider as potable water.

Sanitary Tests for coliforms

The standard multiply tube fermentation technique (most probable

number M.P.N.) are used for coliform detection.

(This method involves 3 routine standards tests):

1) The presumptive test.

2) The confirmed test.

3) The complete test.

1. Presumptive test

A serial dilution of fermentation tubes (macConkey broth),are

inoculated with water sample. These tubes are incubated for 24 - 48hr at

37ºC.

Procedure

1. Make serial dilution (10-x

) of water samples as the following:

Sewage water 9 dilutions

River water 8 dilutions

Stagnant water 6 dilutions

Brooklet water 5 dilutions

Tank water 4 dilutions

Drinking water 3 dilutions

2. Suspend 1 ml of water sample in 9 ml D.W.(10-1

),then transfer 1 ml of

first dilution to another tube to prepare the second dilution(10-2

).

3. Prepare the last 3 dilutions for each water sample in macConkey

broth tubes rather than D.W.

ex…. if the water sample require 5 dilutions (Brooklet water), the 1st and

the 2nd dilutions use D.W. , while the 3 last dilutions use macConkey

broth (suspending 1 ml of 2nd dilution in 9 ml macConkey broth (10-3

)

and the other two remaining dilutions prepare in same way(10-4

and 10-5

).

3

4. Incubate MacConkey tubes for 24hr at 37ºC.

5. read the result as the following:

(+ve)….. coliform change the color of macConkey broth from red to

pink due to lactose fermentation ( as macConkey broth contain neutral

red indicator. change to pink in acidic environment).

6. Enumerate +ve test tubes for each P1,P2 and P3 .then find the MPN

value of coliform by using MPN standard table .

The number of coliform present is not absolute, but is a statistical

estimate.

2. Confirmed test

The positive tubes in Presumptive test does not mean that fecal coliform

are detected? other coliforms also can give false positive presumptive

test because they also can ferment lactose. Therefore a confirmed test is

necessary to identify the presence of fecal coliform.

mid reverse of ×No. of cells / ml =M.P.N.value

×? dilution

4

Procedure

Transfer 1ml of any +ve tube of presumptive test to another

macConkey broth tubes (contain Durham tubes) incubate for 24hr

at 44.5ºC.

+ve result…… for fecal coliform: color change to + gas in durham

tube.

3. Complete test

The complete test is use to be ascertain about the presence of fecal

coliform in water.

Procedure

1. Streak a loop full from the +ve tubes on macConkey agar or Eosin

Methylene Blue agar (EMB).

2. Incubate plates at 37ºC for 48hr.

3. Macroscopically ……..coliform colonies on macConky agar are

(pink with dark center and opaque) while on EMB agar (colonies

nucleated with metallic sheen).

4. Make a slide from colony on glass slide with Gram stain.

1

Aquatic microbiology lab (8):

Feacal Bacterial Indicators

There are other groups of bacteria invariably present in human and animal feces:

2- Clostridium especially Clostridium perfringens

3- Fecal streptococci, especially Streptococcus faecalis

4- Certain species of anaerobic bacteria, Bifidobacterium bifidus.

These species are easily isolated from water by the use of relatively simple methods of

selective cultivation and are readily identified. The first two species are used frequently as

indices of fecal pollution in both water and foods.

2-Clostridium

Clostridia… are obligate anaerobic, gram-positive, endospore-bearing , large rods ,

with rounded ends. The majority of Clostridia are harmless and helpful saprophytes . some

of them live in intestinal tract of human and animal, other cause serious diseases of human

and animals.

The advantage of testing the presence f Clostridia especially Clostridium perfringens

pollution, because the old fecal an :type of pollutionthe inguggest, is sin water samples

survival time of pollution indicator organisms in water ( Clostridium perfringens,

resistant spores can survive in water).

suggests E. coli,, particularly bacteria in the absence of the otherspresence of this The

both , but if (old pollution) long time considerablefor a presence d pollution that may ha

recent ( a few hours or dayssuggest relatively founded in water ) E. coli &(Clostridia

pollution) .

(1) Presumptive test

1. Full a glass tube of tested water sample without any serial dilution ( heat the tube in

water bath for 10 min at 80 °C ?).

2. Transfer 1ml of heated water sample to tubes with selective medium differential

reinforced Clostridia medium (DRCM) supplemented with sodium sulfate and

ferrous citrate in equal volumes to great reducing state , incubate tubes an aerobically

(anaerobic gar) at 37 °C for 48 hr.

3. +ve tubes detected by the formation of black deposit of ferrous sulphate FeS .

2

(2) Confirmed test

The confirmed test is done to confirm the presence of Clostridium perfringens in

positive tubes by using Litmus Milk ( stormy fermentation phenomenon), since

Clostridium perfringens ferment sugar in litmus milk(lactose to lactic acid) lead to greats

extremely acidic condition which lead to coagulation of milk protein(Casein) and the gas

that formed through sugar fermentation push protein clot to the surface.

Procedure

1. Transfer 1ml of +ve tubes to preheated test tubes filled with litmus milk medium

(precaution: be carefully do not expose samples to air).

2. Incubate tubes an aerobically at 37° C for 48 hr.

3. Detect +ve tubes for stormy fermentation.

4. Make a slide, stain with gram stain and examine under oil immersion.

1

Aquatic microbiology Lab (9):

3-Fecal Streptococci The fecal streptococci found in the feces of humans and other worm- blooded animals:

(a)- Streptococcus faecalis

(b)- Streptococcus bovis

(c)- Streptococcus equines

very the intestinal tract their presence indicates limited survival time outside Because of

recent pollution.

Fecal streptococci data verify fecal pollution and many provide additional information

combination with data on nI ,pollutionprobable origin of and recencyconcerning the

coliform.

(FC/ FS ) Ratio

The relation of the fecal coliform and fecal streptococci density may provide information

on the potential source of contamination, (N. of Fecal coliform /N. of Fecal streptococci

while if the ,omestic wastes )fecal pollution ( d humandicates in greater than 1ratio if the, )

pollution . blooded animals –worm indicates less than 1ratio

(M.P.N. ) of fecal streptococciMost probable number

Presumptive test -1

Procedure:

Azide ml of 4.5 withsterile tubes -of water ) to 3 typesml of water (different 0.5 add -1

) .1

-10mixed well ( dextrose broth

2-Transfer 0.5 ml from previous tubes to another 3- tubes of Azide dextrose broth mixed

well (10-2).

2

3- Finally transfer 0.5 ml from (10-2) to another 3- test tubes with Azide dextrose broth

and mixed well (10-3).

Now we have 9-tubes contain Azide dextrose broth (10-1), (10-

2), (10-

3).

because this bacteria do not grow ?sfew number of dilutionThe procedure include a : Notes

easily in media.

4- Incubate at 370 C for 24-72 hr.

5- Record the results if the tubes negative or positive ( +ve: turbid + change in color from

.yellowto purple

6- calculate the number of cells for 1 ml (cell /ml) by using the value of (M.P.N.) from the

table .

7- Compare the cell number of coliform(previous data) with the cell number of

streptococci, to evaluate the ratio of (FC/ FS).

Confirmed test-2

Confirmed test for streptococci is essential to Confirm the the presence of fecal streptococci

especially Streptococcus faecalis .

Procedure:

1- Transfer 0.5 ml from +ve results of Azide dextrose broth (yellow tubes) to another

tubes contain 4.5 ml of Azide dextrose broth, mixed well.

2- Incubate the tubes at 450 C for 48 hr.

will confirm the presence yellowto purple from change color ty andirbid+ ve result ….tu -3

of Streptococcus faecalis

testComplete -3

.

Inoculate the selective solid media for

Streptococci Pfiezer selective agar (PSE

agars ) with positive result of Confirm test .

colonies of Streptococcus faecalis on this

brown color encircled by -blackmedium have

1

Aquatic microbiology Lab (10):

Detection of pathogenic bacteria

Pathogenic bacteria come into the water mainly from domestic waste water and most of

them cannot stay for a long time because soon they die, so the water-laden feces (Sewage)

are the source of disease particularly pathogenic intestinal microorganisms.

The study and investigation of coliform bacteria is important for its close relationship with

pathogenic bacteria, it is possible to isolate pathogenic bacteria in the case of presence of

coliform bacteria in water , so this test is important always. But we will depend on previous

. i.e pathological types of bacteria to other and investigatedtests have made to the coliform

Salmonella , Shigella , Vibrio .

Isolation and Identification of Salmonella and Shigella

Salmonella

G-ve bacteria, bacilli, non- spore former, motile by peripheral flagella, mostly non lactose

fermented, produce H2S.

by:Enterobacteriaceae Distinguishing from other

1- Motility

2- Do not produce capsule

3- non-lactose fermenter through 18-24h when grow on Macconky or S.S.agar so they have

.pale colonie

Shigella

G-ve bacteria, bacilli, non- spore former, non-motile, non-lactose fermenter, do not produce

H2S.

2

Shigellafrom Salmonella Characterized

1- Production of H2S.

2- Produce gas and acidity in sugar solution.

3- Motile

4- Most of them do not have the ability to produce Indol .

dealing with contaminated food and whenof salmonellosis ncaused infectio…. Salmonella

drink.

can cause dysentery through contaminated food and drink when it Shigella….. Shigella

moves to the large intestine and begins to reproduce, the dysentery accompanied by diarrhea

with blood and mucous materials, abdominal pain and neurological disorders.

.. Enrichment

: athogenic bacteria, becausepThe important steps to isolate the

Pathogenic bacteria are few in number in the water - 1

bacteria.athogenic p to media toxicselective ften theO -2

Procedure:

taken are ifferent water samplesD -1

enrichmentof volume equal with media enrichmentAdd the water sample to multiplier -2

.media

Shigella and Salmonella media for Enrichment

1- Selenite broth

ed within incubatshould be and it Salmonellaof the rapid proliferation allowsThis medium

.by turbidity and orange color indicated growth hours; the of 24 period a

3

: Notes

the growth of coliform bacteria, sallowof selenite broth the incubation period Increasing

.the early hours of incubation in this medium inhibited duringwhich were

broth GN -2

and fecal , it inhibited coliform Shigella This medium gives a good growth of the

of the growth sallow more than 24 hours isiod , but if the incubation per streptococci

.Pseudomonus aeroginosa, Proteuslike -bacteriaacidophilic

media. turbidity formed in the indicated by The growth Note:

in Selective media Growth

: should do the followingbacteria we athogenicp-nonathogenic bacteria from pTo separate

pathogenic for appropriate should be he temperatures and duration of incubationT -1

bacteria.

appropriate media. Use -2

solid selective media: Classification of

medium1) Differential (

microorganisms othermaterials inhibit the growth of contain This media contain or does not

but contains materials intended to give the bacteria special it,unintended to grow on

allowing for agar)(EMB bacteria, such as from otherdistinguish them characters that

give the E. coli but, only E. coliand genus of bacteria other than the of typesgrowth

other bacteria appear in red color. ,sheenmetallic green phenomenon of

2 ) Selective media )

the growth of bacterial groups without the smedia contains inhibited materials, allow This

other, but not necessarily with material distinguish these groups from each other, so it could

for example:media at the same time be selective and differential

medium because it contains substance selective as consider is media hisT …EMB agar -1

bacteria and it is ve -bacteria and allows the growth of G ve+ the growth of G ed inhibit

from the other enteric bacteria . E. coliit distinguishes the media because differential

; it is selective and differential medium as consider is his mediaT …acconky agarM -2

the growth of inhibit bile salt whichand the crystal violet selective because it contain

etween distinguish bve enteric bacteria ,also it -the growth of G sand allow bacteria veG+

lactose -while nonpink colonies appear as lactose fermenter bacteriave -the G their genus,

.pale coloniesfermenter bacteria appear as

4

agar ) Shigella -Salmonella S.S. agar (-3

bile it contains the because selective and differential medium, This media is consider as

also contains lactose to differentiate and itbacteria ve+ G salts which inhibit the growth of

Shigella and Salmonella bacteria , ctose fermenterla -and non fermenterbetween lactose

fermented lactose sugar also them cannotcolonies appear as pale colonies because both of

S 2of Hproduce Salmonella , Shigella and Salmonella this medium distinguishes between

which is concentrated in the )precipitation which reacts with iron, giving the FeS (Black

pale colonies without a black colonies appear as Shigellawhile center of the colonies

. , ,this media contain Neutral red as indicatorS2H ter because it is not produce ofcen

Procedure:

while the growth on Selenite broth turbida as appears GN broth growth onthat Notes -1

.orangechanged to coloras well as the appears as a turbid

dish is divided into two equal halves by which the first -the Petriand is prepared S.S.agar -2

while the second half is color) (turbidity and orange Selenite broth from half is inoculated

.(turbidity)GN broth from inoculated

for 24h.˚Incubate the plate at 37C -3

.colonies appearance of Notes the -4

SalmonellaPale colonies + FeS -5

Shigella without FeSPale colonies

Make a slide and stained with Gram stain -6

the microscope under Shigellaand Salmonella distinguish between cannotWe :Notes

former.spore -non bacilli,, ve-features; Gbecause they have same